Use of the anionic dye carboxy-2 ',7 '-dichlorofluorescein for sensitive flow cytometric detection of multidrug resistance-associated protein activity

Multidrug resistance-associated protein (MRP) and P-glycoprotein are drug e fflux pumps conferring multidrug resistance to tumor cells and sharing nume rous substrates. In order to determine a flow cytometric assay allowing to analyse MRP activity in cancerous cells in a sensitive and specific manner, cellular accumulation and efflux of the anionic fluorescent dye carboxy-2' ,7'-dichlorofluorescein (CDF) were studied by flow cytometry using mainly M RP-overexpressing lung GLC4/Sb30 cells and parental GLC4 cells. GLC4/Sb30 c ells were found to display reduced accumulation and enhanced efflux of the dye when compared to their parental counterparts. Probenecid, a well known blocker of MRP, strongly enhanced CDF accumulation in GLC4/Sb30 cells throu gh inhibiting efflux of the dye; it also increased CDF levels in GLC4 cells , although to a lesser extent, which may likely be linked to the low, but d etectable, expression of MRP in these cells. Comparison of CDF retention wi th that of calcein demonstrated that the former dye was the most efficientl y effluxed by GLC4/Sb30 cells. In contrast to MRP overexpression, that of P -glycoprotein was not found to alter cellular CDF labelling whereas it stro ngly impaired calcein staining. These results indicate that CDF is a substr ate for MRP, but not for P-gp, which may likely be useful for sensitive and specific flow cytometric determination of MRP activity in clinical samples .